Endocannabinoid tone versus constitutive activity of cannabinoid receptors

Abstract

This review evaluates the cellular mechanisms of constitutive activity of the cannabinoid (CB) receptors, its reversal by inverse agonists, and discusses the pitfalls and problems in the interpretation of the research data. The notion is presented that endogenously produced anandamide (AEA) and 2-arachidonoylglycerol (2-AG) serve as autocrine or paracrine stimulators of the CB receptors, giving the appearance of constitutive activity. It is proposed that one cannot interpret inverse agonist studies without inference to the receptors' environment vis-à-vis the endocannabinoid agonists which themselves are highly lipophilic compounds with a preference for membranes. The endocannabinoid tone is governed by a combination of synthetic pathways and inactivation involving transport and degradation. The synthesis and degradation of 2-AG is well characterized, and 2-AG has been strongly implicated in retrograde signalling in neurons. Data implicating endocannabinoids in paracrine regulation have been described. Endocannabinoid ligands can traverse the cell's interior and potentially be stored on fatty acid-binding proteins (FABPs). Molecular modelling predicts that the endocannabinoids derived from membrane phospholipids can laterally diffuse to enter the CB receptor from the lipid bilayer. Considering that endocannabinoid signalling to CB receptors is a much more likely scenario than is receptor activation in the absence of agonist ligands, researchers are advised to refrain from assuming constitutive activity except for experimental models known to be devoid of endocannabinoid ligands.

Figure 1

The G protein activation cycle, showing the opposite effects of a traditional agonist (Ag) and an inverse agonist (Inv Ag) on G protein activation and de-activation. (A) Agonist-dependent activation, by which the agonist serves as a catalyst that promotes the dissociation of receptor and G protein heterotrimer (high affinity for GDP) into the active components (Gα has a high affinity for GTP). (B) Constitutive activation, in which G protein activation occurs in the absence of agonist. In this scenario, an inverse agonist can stabilize the inactive receptor–G protein complex, further increasing its affinity for GDP and decreasing affinity for GTP, and prevents constitutive activation of G proteins.

Figure 2

Pathways for 2-AG metabolism (2A), putative pathways for AEA synthesis (2B) and AEA breakdown (2C). (A) 2-AG is formed in step 1 by the action of DAG lipase upon DAG and 2-AG is metabolized in step 2 by MAG lipase, ABHD6 and ABHD12 to arachidonic acid and glycerol. (B) The metallo-β lactamase, NAPE–PLD, hydrolyses NArPE to form AEA via a one-step reaction (pathway 1). The serine hydrolase, ABHD4, sequentially removes acyl groups from NArPE to form lyso-NArPE and then GP-AEA (pathway 2a–b). The metal-dependant phosphodiesterase, GDE1, hydrolyses GP–AEA to form AEA (pathway 2c). A type-C phospholipase hydrolyses NArPE to pAEA (pathway 3a). PTPN22, SHIP1 or other uncharacterized phosphatases dephosphorylate pAEA to form AEA (pathway 3b). (C) Degradation of AEA to AA and ethanolamine by FAAH. The arachidonic acid moiety is highlighted in red.

Figure 3

Depiction of AEA (orange) carried in the interior of FABP7. The carboxamide oxygen of AEA can interact with FABP7 interior residues R126 and Y128, while the hydroxyl group of AEA can interact with FABP7 interior residues, T53 and R106 (Reggio et al., 2009).

Figure 4

The results of microsecond timescale molecular dynamics simulations of 2-AG interacting with the CB₂ receptor embedded in a POPC bilayer. This figure illustrates the progress of 2-AG from the lipid bilayer into the CB₂ binding pocket as viewed from the extracellular surface of the receptor. 2-AG is located initially in the lipid bilayer surrounding CB₂. The lipid bilayer constituents are not displayed in order to simplify the view. The color scale represents the percentage of the trajectory in which any portion of 2-AG is within 4 Å of residues on CB₂ (defined here as within contact distance). Residues within contact distance are listed on the right and are color coded according to this scale. (A) The 2-AG has partitioned out of bulk lipid and contacts residues in or near the TMH6/7 interface. Highest contact is with F7.35(281) and C7.38(284). (B) 2-AG interaction with residues in the TMH6/7 interface increases with greater than 80% contact occurring with F7.35(281), S7.39(285) and C6.47(257). (C) After 2-AG entry into CB₂, 2-AG begins to contact binding pocket residues on TMH3 (V3.32(113)), TMH6 (W6.48(258)), TMH7 (C7.42(288)) and the EC-3 loop (D(275)). (D) Subsequent to protonation of D3.49 and D6.30, 2-AG contacts multiple residues on TMH3/6/7 and the EC-3 loop with formation of hydrogen bonds with D(275) in the EC-3 loop and to a lesser extent with S7.39(285) (Hurst et al., 2010).